Field
The present invention refers to the field of virology, more precisely to methods for generating Hepatitis E Virus (hereinafter HEV) stocks. Specifically, the present invention discloses methods for propagating and titrating high titer HEV stocks.
Description of the Related Art
HEV (genus Hepevirus, family Hepeviridae) is a small non-enveloped/pseudo-enveloped virus, with a single-stranded positive-sense, polyadenylated RNA genome of approximately 7.2 kb. There are four genotypes of HEV that have been identified, but only one serotype. Genotypes 1 and 2 are mainly responsible for waterborne infections in underdeveloped countries and cause disease primarily in humans and higher primates. Infections are typically self-resolving and acute, lasting at most 2 to 7 weeks, but may be fatal especially in pregnant women. Genotypes 3 and 4 are associated with endemic (autochthonous) infections in industrialized countries. These two genotypes cause disease largely in swine but humans can become accidental hosts as a result of food or zoonotic exposure. Clinical disease is typically asymptomatic and mild in young adults, but can become clinically apparent in older men. In addition genotype 3 and 4 infections can become chronic in immune-suppressed persons, such as organ-transplant patients or AIDS patients.
Recently hepatitis E has been categorized as a transfusion transmittable infectious disease. Given the worldwide spread of HEV in recent years, concerns have been raised regarding the safety of blood- and plasma-derived products. The virus safety profile of blood- and plasma-derived products can be assured by performing clearance studies that demonstrate the virus reduction and/or clearance capacity of their manufacturing processes. During these clearance studies a known amount of virus is deliberately spiked into a blood or a plasma product intermediate and then the spiked material is processed using a bench scale model of the manufacturing process. Virus reduction and/or clearance across a step is determined by comparing the amount of virus before and after treatment.
Virus clearance studies require large quantities of high titer virus and the lack of an efficient cell culture system for HEV has hampered the ability to perform such studies for HEV. Several HEV cell culture systems have recently been developed to address this problem.
A genotype 3 and a genotype 4 strain were adapted by Okamoto and colleagues to grow in A549 human lung cells reaching HEV RNA titers of 3.9×108 copies/mL. In addition, said genotype 4 strain was also cultured in PLC/PRF/5 cells (human hepatoma cells) but with lower titers.
A second genotype 3 (strain Kernow-C1) was adapted by Emerson and colleagues to grow in HepG2/C3A human hepatoma cells obtaining a titer of 4.61×108 genomes/mL after 6 passages. Studies showed that the adaptation for growth in vitro resulted after the acquisition of 174 ribonucleotides of the S17 human ribosomal protein gene. Because genomes with the same insertion were detected in the original virus inoculum, a fecal suspension from a chronically infected HIV-1 patient, the recombination/insertion event had occurred naturally and was not an artifact of cell culture. Attempts to grow said strain in PLC/PRF/5 and A549 cells were unsuccessful or resulted in lower titers, but the virus infected and replicated in kidney cells from swine, the major zoonotic host for genotype 3 viruses.
Human hepatoma cells are difficult to grow and may require special cell plating methods including the use of coatings such as collagen, fibronectin, gelatin and/or poly-L-lysine to facilitate cell attachment and/or cell growth. In addition, not all of these coatings work well for all cell types.